Effect Of Protein Nitrosylation On Calpain Activation And Protein Proteolysis Of Pork During Postmortem Aging

Fresh meat quality including meat tenderness and water holding capacity are important traits for consumer acceptance and industry profitability in China.Reactive oxygen species and reactive nitrogen species are accumulated in postmortem muscles of pork.Reactive nitrogen species includes nitric oxide(NO)which is catalyzed by nitric oxide synthase(NOS)from L-arginine to L-citrulline.Protein nitrosylation,covalent attachment of nitric oxide to sulfur of cysteine,could alter protein conformation and function.Protein degradation caused by calpain during postmortem aging could contribute to the variation of water holding capacity of pork.As a cysteine protease,the active sulfur of calpain could be attacked by the reactive substances to affect its activity and autolysis.Therefore,the research is to investgate protein modification of nitrosylation,especially the effect of calpain activity and conformation,and subsequently the myofibril degradation to alter the formation of meat quality during pork postmoterm aging.1.Effect of nitric oxide on ?-calpain activation,protein proteolysis and protein oxidation of pork during postmortem agingThe aim of the current research was to examine the influence of nitric oxide on calpain activation,protein proteolysis and oxidation in post-mortem pork.Nitric oxide synthase inhibitor(L-NAME)and NO enhancer(DETA/NO)were used to regulate the levels of NO.SDS-PAGE and Western Blotting were used to detect the autolysis of calpain and the degradation of protein.Protein oxidation was investigated by the content of carbonyl and free thiol.Results showed that the autolysis of ?-calpain increased by the treatment of NOS inhibitor(P<0.05).NOS inhibitor significantly increased the levels of titin and nebulin degradation.Higher levels of protein oxidation were observed after samples incubated with NO donor than treatment of NOS inhibitor(P<0.05).These data indicated that NO could participate in regulating calpain activation and its proteolysis activity during post-mortem aging.2.Influence of ?-calpain nitrosylation on its autolysis and myofibrillar protein degradation of pork in vitroThe objective of this research was to investigate the effect of protein nitrosylation on the degradation of myofibrillar protein of pork in vitro and ?-calpain autolysis.After reacting with different levels of NO(GSNO),?-calpain was incubated with myofibrillar protein of pork.SDS-PAGE and Western blotting were used to examine calpain autolysis and protein degradation.Biotin Switch method was used to identify the level of calpain nitrosylation.The calpain autolysis decreased as the concentration of GSNO increased.The results also showed that the degradation of titin,nebulin,desmin and Troponin-T decreased after calpain incubating with higher levels of GSNO.Different levels of GSNO could lead to different levels of calpain nitrosylation.With the levels of NO increasing,the level of calpain nitrosylation improved.These results demonstrate that nitric oxide could lead to calpain nitrosylation which inhibits the autolysis of calpain and its proteolysis activity.